Potato dextrose agar (pda)

The specimens of Rubia tinctorum under study were collected on the banks of the Carrión river as it passes through the town of Palencia (Spain). The roots were shade-dried and pulverized to fine powder in a mechanical grinder. Samples from different specimens (n = 25) were thoroughly mixed to obtain composite samples.
Chitosan (CAS 9012-76-4; high MW: 310,000–375,000 Da) was supplied by Hangzhou Simit Chem. & Tech. Co. (Hangzhou, China). Neutrase^TM 0.8 L enzyme was supplied by Novozymes A/S (Bagsværd, Denmark). Stevioside (CAS 57817-89-7, 99%) was purchased from Wako Chemicals GmbH (Neuss, Germany). Quantities of 4-tert-butyl-2-phenylphenol (CAS 98-27-1, 97%), 1,2-dihydroxyanthraquinone (CAS 72-48-0, 97%), sodium alginate (CAS 9005-38-3), calcium carbonate (CAS 471-34-1, ≥99.0%) and methanol (CAS 67-56-1, UHPLC, suitable for mass spectrometry) were acquired from Sigma-Aldrich Química (Madrid, Spain). Agar (CAS 9002-18-0) and PDA (potato dextrose Agar) were supplied by Becton Dickinson (Bergen County, NJ, USA).

Microbial strains used in this study and growth media are listed in the Supplementary Table S2 and Table S3, respectively.
The propagation of vectors and plasmids was carried out in E. coli DH5α, grown in lysogeny broth medium (LB), supplemented with 100 µg/mL ampicillin. Yeasts were grown on potato dextrose agar (PDA, Becton Dickinson) or on solid yeast extract peptone dextrose medium (YPD) at 37 °C.
For the generation of conidia, P. chrysogenum was cultivated on solid defined minimal medium (MM) at 25 °C, N. crassa on Vogel’s agar plates at 37 °C under continuous light and A. fumigatus, A. niger and A. terreus were grown on PDA (Becton Dickinson) at 37 °C. T. rubrum was grown on oatmeal agar. Conidia were harvested in spore buffer (0.9% NaCl (w/v), 0.01% Tween 80 (v/v)). For transformation, 10⁸ conidia of P. chrysogenum Δpaf^{21 (link)} were grown in 200 mL CM for 38 h at 25 °C under continuous shaking (210 rpm). Conidia (1–2 × 10⁸) of P. chrysogenum Q176 and P. chrysogenum pafB were cultivated in 200 mL MM for 24–144 h at 25 °C under continuous shaking for Northern, Southern and Western blot analyses and PAFB production, respectively. PAFB single (¹⁵N) or double (¹⁵N/¹³C) isotope-labelling for NMR analysis was carried out by replacing the nitrogen and/or carbon source in MM by 0.3% Na¹⁵NO₃ and/or 1% ¹³C-glucose (w/v) (Euriso-Top), respectively.

S. sclerotiorum hypovirulent strain DT-8 carrying SsHADV-1 (CCTCC M 2019328) was isolated from a sclerotium formed on a diseased stem of rapeseed from Hunan Province, China. The virulent SsHADV-1-free strain, DT-8VF (VF means virus-free), was derived from strain DT-8 by hyphal-tip isolation [36 (link)]. Both strains were grown on potato dextrose agar (PDA, Becton, Dickinson and Company, Sparks, MD, USA) plates at 20 °C, and stored on PDA slants at 4 °C.